Abstract

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy usually refractory to current treatment strategies and associated with short overall survival. By applying next-generation functional testing of primary patient-derived lymphoma cells using a library of 106 US Food and Drug Administration (FDA)-approved anticancer drugs or compounds currently in clinical development, we set out to identify novel effective treatments for T-PLL patients. We found that the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax (ABT-199) demonstrated the strongest T-PLL–specific response when comparing individual ex vivo drug response in 86 patients with refractory hematologic malignancies. Mechanistically, responses to venetoclax correlated with protein expression of BCL-2 but not with expression of the BCL-2 family members myeloid cell leukemia 1 (MCL-1) and BCL-XL in lymphoma cells. BCL-2 expression was inversely correlated with the expression of MCL-1. Based on the ex vivo responses, venetoclax treatment was commenced in 2 late-stage refractory T-PLL patients resulting in clinical responses. Our findings demonstrate first evidence of single-agent activity of venetoclax both ex vivo and in humans, offering a novel agent in T-PLL.

Introduction

T-cell prolymphocytic leukemia (T-PLL) is an aggressive T-lymphoid malignancy characterized by proliferation of postthymic prolymphocytes.1,2 Patients typically present with elevated and exponentially rising lymphocyte counts along with splenomegaly, hepatomegaly, lymphadenopathy, and effusions.1 Responses to alkylating agents or polychemotherapy are poor with a median survival of just 7 months.2 The use of the monoclonal anti-CD52 antibody alemtuzumab has improved response rates to 75% or even higher when applied in the first-line setting.3⇓⇓-6 The combination of chemotherapy regimen fludarabine, mitoxantrone, cyclophosphamide (FCM) followed by alemtuzumab increased responses to 90%.7 However, despite high response rates all patients eventually relapse with a median progression-free survival of <12 months.

Study design

Patients and ex vivo drug screen

Samples were obtained and processed after consenting under protocols EKNr1830/2015 and EKNr2008/2015 approved by the local ethics commission. Venetoclax was provided by AbbVie as part of a single-patient preapproval access program. All patient samples (bone marrow aspirates, peripheral blood, or excised lymph node) were prepared freshly and used for drug-screening analysis within 3 hours of harvest. Resulting single-cell suspensions (in RPMI 1640 plus 10% fetal calf serum) were seeded at 1 × 105 cells per well in 384-well plates containing compound libraries with each drug in 4 concentrations in triplicate (supplemental Table 1, available on the Blood Web site). Drug-concentration range was determined by dose-response experiments in primary cells and cancer cell lines for each compound. Differential cell viability was determined after 72 hours with CellTiter-Glo (Promega) on an EnVision plate reader (PerkinElmer). Dose-response curves and immunoblotting were performed on viably frozen samples. Viability data of these samples after thawing was determined by the CASY Cell Analyzer (OLS) (supplemental Figure 3).

Tissue microarray

Data calculation and statistics

Drug responses normalized as percentage of controls were calculated as areas under the curve (AUC) as the sum of all data points for a respective drug in individual patient samples.17P values were calculated using Wilcoxon, Mann-Whitney, and Kruskal-Wallis tests as indicated.

Acknowledgments

The authors are grateful to their patients for donating samples and clinical data for this study. The authors thank AbbVie (Emma Ariola, Alexander Dorr) for providing venetoclax, and Elisa Cerri and German Pena for critically reading the manuscript.

This work was supported by Austrian Science Fund (FWF) grants P27132-B20 (P.B.S.), F4701-B20 (S.K.), SFB F4707 (R.M.), and SFB-F06105 (R.M.); the Austrian Federal Ministry of Science, Research and Economy and the National Foundation for Research, Technology and Development (S.K.); and the Anniversary Fund of the Oesterreichische Nationalbank (OeNB) grant P15936 (P.B.S.).

Footnotes

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

. Development and validation of a single-cell network profiling assay-based classifier to predict response to induction therapy in paediatric patients with de novo acute myeloid leukaemia: a report from the Children’s Oncology Group. Br J Haematol. 2013;162(2):250-262.